HCV are recognized respiratory pathogens that can cause as much as 30% of common colds (33
). However, some studies suggest their possible neurotropism. Human neural cell lines and primary cultures of human brain cells are susceptible to HCV-229E infection (1
). Yeager et al. (51
) have shown that hAPN (CD13) acts as a cellular receptor for infection of human lung fibroblasts. However, CD13 has been found on synaptic membranes, but not on glial cells of the human brain (27
). Since virus tropism results in part from the nature, the number, and the distribution of cellular receptors, we asked whether CD13, which is present at the surfaces of many cells (2
), was indeed present at the surfaces of human neural cell lines and, consequently, whether strain 229E of HCV uses this protein for attachment and infection.
By flow cytometry, we were able to show expression of CD13 on cell lines of neuronal, astrocytic, and oligodendrocytic phenotypes but not of the microglial phenotype. Compared to the neuronal cell lines, the GL-15 astrocytic cell line showed an intermediate level of expression of CD13, which was nonetheless higher than that observed with the other two astrocytic cell lines, U-87 MG and U-373 MG. This might explain our previous observation that the latter two astrocytic cell lines require a higher MOI for infection compared to the GL-15 cell line. Pig astrocytes were previously shown to express APN (4
), but our results give the first report of APN expression on human astrocytes and oligodendrocytes. The absence of CD13 on the microglial cell line CHME-5 is surprising, because this protein is known to be present on macrophages (27
). This result might be a consequence of the transformation of this cell line following its transfection with the simian virus 40 large T antigen (20
). Nonetheless, this absence is consistent with the implication of CD13 as a cellular receptor on human neural cell lines since the CHME-5 cell line is not susceptible to HCV-229E infection (data not shown). Unfortunately, no other human microglial cell lines are to our knowledge available at this time, and further studies of HCV-229E infection of microglial cells will require either CD13 transfection into CHME-5 or the production of other immortalized cell lines.
Having shown that CD13 is indeed expressed on human neural cells, the next step of this study was to verify whether this protein acts as a cellular receptor for HCV-229E infection of human neural cell lines. APN has been found to be a cellular receptor for HCV-229E infection of human lung fibroblast cell lines (51
), but there had previously been no direct proof of the use of CD13 by HCV-229E for infection of human neural cells. Presumably, this virus could have used an alternative receptor, as has been shown for MHV (52
). To evaluate the role of CD13 in infection of human neural cell lines by HCV-229E, we used a CD13-specific MAb to inhibit attachment of HCV-229E onto human neural cell lines and to inhibit infection of these cell lines by HCV-229E. The anti-CD13 MAb WM15 was able to substantially diminish attachment of HCV-229E to all human neural cell lines tested, except for the CHME-5 microglial cell line. The fact that WM15 did not recognize HCV-229E indicated that the inhibition seen was not a consequence of the association of this antibody with the virus that would block its attachment to the cells. Also, the inhibition did not appear to be caused by nonspecific attachment of antibodies to HCV-229E, since nonneutralizing anti-S antibodies against HCV-229E failed to inhibit HCV-229E attachment. Therefore, it seems that the presence of CD13 at the cell surface is necessary for the specific binding of HCV-229E to human neural cell lines of neuronal, astrocytic, and oligodendrocytic phenotypes (and presumably microglial cells also). These experiments also showed that virus binding was variable, depending on the cell lines used (H4 > SK-N-SH > MO3.13 > GL-15 > U-373 MG > U-87 MG). The correlation that was observed between the levels of expression of CD13 and HCV-229E binding to human neural cell lines seems to indicate that a higher-level expression of CD13 is accompanied by the binding of more viruses to these cells. This could explain our observation that a higher MOI is required for infection of some neural cell lines. However, an overexpression of CD13 does not necessarily translate into a more-efficient infection; overexpression of porcine APN at the cell surface results in a lower level of TGEV replication (17
), a reduction that could be caused by the binding of porcine APN to the viral S glycoprotein, which apparently hindered virus maturation.
We also evaluated the role of CD13 in the initiation of a productive HCV-229E infection of human neural cell lines. Neuronal, astrocytic, and oligodendrocytic cells pretreated with the anti-CD13 MAb WM15 showed a marked decrease in the extent of HCV-229E infection. Like the inhibition of attachment experiment, control antibodies did not decrease the level of infection compared to cells untreated with MAb.
Overall, these inhibition of attachment and infection experiments suggest that, like human lung fibroblastic cell lines, CD13 acts as the cellular receptor for HCV-229E infection of human neural cell lines. It remains to be determined how and if HCV-229E could gain access to the brain to infect human neural cells. One possibility might be via the pericytes at the blood-brain barrier. The presence of CD13 on these cells (25
) might provide an entry pathway for HCV-229E to the CNS. Indeed, primary cultures of human endothelial cells are susceptible to HCV-229E infection (12
). This infection might be the initial step for the propagation of HCV-229E to the CNS via infection of astrocytes, which are cells that are susceptible to HCV-229E infection (7
Other coronaviruses, such as TGEV, FeCV, and FIPV, and human cytomegalovirus use APN for infection (15
). The high levels of expression of this protein on the surfaces of epithelial cells of the respiratory and digestive tract could presumably confer on this protein a receptor function for several viruses. Therefore, it is possible that other viruses use APN as a cellular receptor, but that does not necessarily mean that the same region of APN might be recognized by different viruses. The anti-CD13 MAb WM15 used in this study was previously shown to block the enzymatic activity of CD13 (51
), and we now show that it prevents HCV-229E infection of human neural cell lines. Also, molecules chelating the zinc ions required for CD13 enzymatic activity were shown to inhibit HCV-229E infection, but other molecules, such as bestanin and actinonin, which bind to the active site of CD13, were incapable of preventing infection by this virus, unlike human cytomegalovirus (41
). Therefore, it seems that HCV-229E recognizes an epitope near the catalytic site of CD13. A recent report showed that a small region within the amino-terminal part of the feline APN is essential for HCV-229E infection (24
Expression of CD13 on neurons, astrocytes, and oligodendrocytes would explain their susceptibility to HCV-229E infection. In addition, CD13 is a protein expressed on the surfaces of many cell types. This suggests that these cell types might be susceptible to HCV-229E infection. However, only human lung fibroblasts and CNS cells have so far been shown to be susceptible to HCV-229E infection (7
). A complex relationship could exist between viral tropism and expression of CD13. For example, the poliovirus receptor is present on many cell types, even those not susceptible to poliovirus infection, suggesting that the presence of a coreceptor may be necessary for infection of permissive cells (40
). Indeed, recent studies suggest that other postattachment factors might be necessary for poliovirus replication since CD44, a potential cellular receptor for this virus, is not required for poliovirus infection (8
). The presence of a coreceptor for HCV-229E has not been described and may not be required, since we showed an apparently monophasic attachment of radiolabeled HCV-229E onto L-132 cells. However, such a putative coreceptor might be necessary only for virus penetration into the cell, something that cannot be detected by the above experiment. Similarly, even though transfection of CD13 into murine cells was sufficient to render these cells susceptible to HCV-229E infection (51
), the possibility that these cells already expressed the putative coreceptor at their cell surface cannot be excluded. Nevertheless, our results do indicate that CD13 is essential for infection of the neural cell lines tested.
The results of our study are consistent with the possibility that CD13 plays an important role in the replication cycle of HCV-229E in the CNS, and its expression and use are consistent with the suspected neurotropism of this virus. Obviously, a better understanding of the role of this molecule will surely help the development of treatments to prevent human infections. This study relied on continuous human neural cell lines that may lose or gain functions during immortalization, although previous studies have shown a nice correlation between in vitro and in vivo coronavirus replication data (for example, see reference 34
). Nevertheless, it will be interesting to verify the expression of CD13 on primary cultures of human neural cells and on human brain sections. The present study provides a strong rationale for such experiments aimed at the characterization of the neurotropism of human coronaviruses and their possible involvement in neurological diseases.