To determine a role for C-type lectins in the entry of IBV into host cells, we transiently expressed either DC-SIGN or L-SIGN in cell lines that are known to be refractory to infection by clinical strains of IBV, such as M41, and examined these cells for virus infection. shows 3T3 (A) or CRFK cells (B) transfected with plasmids expressing DC-SIGN or L-SIGN, or with a control plasmid, and then infected with IBV M41. Cells expressing DC-SIGN or L-SIGN were identified with anti-lectin antibodies. In the absence of lectin expression, we observed no infection with IBV M41. However, in the presence of DC-SIGN or L-SIGN expression, there was a strong correlation of IBV M41 infection in lectin-expressing cells. shows a quantification of the rescue of IBV infection, in this case using 3T3 and CRFK cells lines stably expressing DC-SIGN, as well as 3T3 and CRFK cells expressing feline aminopeptidase N (fAPN)—which has been previously proposed to be a receptor for IBV. Both 3T3 and CRFK cells expressing DC-SIGN were efficiently infected with IBV M41, whereas there was no apparent rescue of infection in 3T3 or CRFK cells expressing fAPN. Overall, these data strongly suggest that over expression of the C-type lectins L-SIGN or DC-SIGN can act as part of an IBV receptor complex and allow infection of 3T3 or CRFK cells that are otherwise resistant to IBV infection, and further support the idea that fAPN is not a functional receptor for IBV.
IBV-M41 infection of 3T3 and CRFK cells is enhanced by introduction of hDC-SIGN or hL-SIGN
IBV-M41 infection is enhanced on 3T3 or CRFK cells stably expressing DCSIGN
To further examine the role of C-type lectins for IBV entry, we treated DC-SIGN-expressing cells with either mannan or anti-DC-SIGN antibodies, and then infected the cells with IBV M41. Mannan is a polymer of mannose that is well recognized to compete with mannose-containing carbohydrates on glycoproteins and block interactions of viruses with C-type lectins. Both mannan and anti-DC-SIGN antibodies inhibited infection by IBV M41 (), further supporting a specific role for DC-SIGN as part of the IBV receptor complex, and indicating that the interactions are mediated through mannose-containing carbohydrate residues present on the viral spike protein. In addition, infection was inhibited by the Ca2+-sequestering agent EGTA (data not shown), further indicating a specific role for C-type lectins, which are known to be Ca2+-dependent for their function.
Specificity of enhancement of infection by IBV-M41
To confirm that DC-SIGN-mediated entry of IBV M41 into cells allowed complete genome replication, we extracted total RNA from either 3T3 or 3T3-DC-SIGN cells infected with IBV M41 (). We then performed RT-PCR to detect the presence of both total and negative-sense viral RNA. In 3T3 cells, we could detect a low level of total viral RNA, but with no detectable negative-sense viral RNA. This indicates that there was some of the original virus inoculum remaining in the sample, but that viral replication had not taken place. In contrast, 3T3-DC-SIGN cells showed a strong signal for both total and negative-sense viral RNA confirming that DC-SIGN expression can rescue replication of IBV M41 in cells that are otherwise refractory to infection.
Replication of IBV-M41 in 3T3-DC-SIGN cells
Sialic acid has been proposed to be part of the receptor complex for IBV M41 (Winter et al., 2006
), and so we examined whether there may be any interplay between sialic acid and C-type lectins for entry of IBV M41 (). 3T3-DC-SIGN cells were infected with IBV M41 in the presence of varying concentrations of neuraminidase, which would cleave sialic acids on the cell surface but not affect C-type lectin function. As a control we used influenza virus, which is well established to use sialic acid as a functional receptor. As expected, influenza infection was strongly inhibited by neuraminidase treatment in a dose-dependent manner. In contrast, there was no overall effect of neuraminidase treatment on IBV M41 infection. These data indicate that there is no functional interplay between sialic acid and C-type lectins as part of the IBV receptor complex, and that C-type lectin-mediated interactions dominate over sialic acid-mediated interactions for IBV M41 infection.
Effect of neuraminidase treatment on 3T3-DCSIGN cells infected by IBV-M41
IBV exists in several serotypes, which are antigenically distinct and so may differ in their receptor requirements. To determine whether C-type lectins can promote entry of a range of different IBVs, we infected 3T3 (not shown) or 3T3-DC-SIGN cells () with the IBV strains Cal99, Conn46, Iowa609, Gray, Iowa97 and JMK, which cover the major virus serotypes. In all cases we observed efficient infection of 3T3-DC-SIGN cells with the IBV strain used, confirming that C-type lectins such as DC-SIGN can promote entry of a wide range of distinct IBV strains.
The 3T3-DCSIGN cell line is susceptible to various IBV strains representing the major viral serotypes
To examine the role of C-type lectin in IBV infection in vivo, we used primary chicken kidney cells, a chicken cell type that is naturally susceptible to IBV infection. While we were not able to inhibit the IBV-M41 strain infection in chicken kidney cells with mannan at 50 μg/ml concentration (data not shown), we observed notable reduction in infection with presence of 0.01M mannose, the sugar monomer enriched in mannan (). As the control, galactose treatment at the same concentration did not render a similar decrease in infection. Our data suggest that there may be a role for a C-type mannose-binding lectin during IBV infection of the chicken host.
Effect of carbohydrates on IBV-M41 infectivity in chicken embryonic chicken cells