Cellular factors modulating the entry efficiency of West Nile virus
West Nile virus (WNV) constitutes a major public health concern considering the changing environment, climate changes, worldwide travel and global trade. An increasing number of outbreaks has been reported for Europe during recent years. Understanding the basic mechanisms of host cell infection will contribute to the knowledge of WNV pathogenesis, transmission and species susceptibility, and help to encounter this global threat in terms of prevention, control and treatment strategies. Identification of receptors and molecules involved in WNV entry is of fundamental importance since the early interaction with the host cell does not only determine cell susceptibility but may also contribute to the exceptionally broad host tropism of WNV. The integrin avb3 had been postulated earlier to function as a receptor for WNV; however, its involvement in WNV entry has been doubted recently. In this context, the present study was designed to clarify the involvement of integrins in WNV entry. Additional questions were addressed as to their role, the extent to which they participate in virus entry, and to possible differences in the binding or entry efficiencies in distinct WNV strains. A cell culture model was established, based on specific integrin knock out cell lines, in order to investigate the susceptibility of these cells to WNV. Wild type, integrin av deficient, b3 deficient and av/b3 double knock out mouse embryonic fibroblasts were isolated from 12.5 days old embryos which possess a specific modification in the particular integrin subunit genomic sequence. The MEF cell lines were cultured and characterised for their integrin expression patterns by immuno fluorescence staining and for their expression levels by flow cytometry analysis. Additionally, integrin b1 deficient mouse kidney fibroblasts and the parental cell line were included in the study. In a parallel approach wild type and integrin deficient cell lines were infected with four WNV strains. Efficiency of binding and of internalisation in terms of virus yields was assayed separately by determination of genome containing virus particle numbers. Furthermore, the involvement of cell associated heparan sulfate as an attachment factor was investigated. The use of two cell types, Lcells (mouse cells from connecting tissue) and CHO K1 (Chinese Hamster Ovary cells) and their glycosaminoglycan or heparan sulfate deficient derivatives allowed comparisons of binding and replication efficiencies. The major conclusions drawn from the experiments are as follows: (i) The presence of either av, b1 or b3 integrins on cell surfaces is not a requirement for a successful WNV infection of the mouse fibroblasts. All four WNV strains were capable to replicate in the integrin deficient cell lines. However, the replication efficiency in b3 deficient cells was substantially lower than in wild type mouse embryonic fibroblasts. Therefore, a rescue of the b3 integrin subunit was introduced into the integrin b3 deficient cell line (refer to results in indent ii, iii) (ii) The hypothesis from the early literature that integrins function as receptors mediating both virus binding and internalisation in an integrated step is rejected. Efficiency of virus attachment to cell surfaces of integrin deficient cells was comparable to that of integrin expressing cells. Moreover, blocking antibodies against b1 and b3 integrin subunits failed to interfere with virus binding to specifically b1 or b3 integrin expressing cells, and had no effect on the infection outcome. (iii) Integrin expression positively affects virus yields as seen in integrin b3 rescue and integrin b1 floxed cells in comparison to the corresponding integrin deficient cell line. At which stage in virus entry or during post entry integrins intervene is not clear. Due to the surface localisation of integrins, a functioning at the level of virus internalisation is suggested, either by mediating endocytosis or by interacting with other unidentified surface receptors in terms of activation or inhibition, or by modification of these proteins, the latter may result in an improved accessibility for the virus. (iv) Differences among the four WNV strains used in this study do not affect the usage of b1 and b3 integrins during entry and/or replication. (v) Rescue of the integrin b3 subunit in CHO K1 cells, and constitutive expression of integrin avb3 does not result in permissiveness of these cells for WNV. Hence, it is assumed that another surface protein is necessary which facilitates virus internalisation, either in cooperation with or independently from integrin avb3. Furthermore, efficiency of binding to CHO K1 cells was essentially the same as seen in other cells. This indicates, in agreement to other findings of the study (see indent ii) that integrin avb3 does neither mediate binding of WNV to the cell surface nor internalisation in a self contained process. (vi) Heparan sulfate serves as a possible attachment factor for WNV but accounts only for a limited portion of virus attachment. The synopsis of these findings strongly suggests avb3 integrins to be involved in WNV entry into target cells. However, results provide evidence that integrins are not the exclusive receptor for WNV, which is in accordance with the multiple receptors concept established in the literature for other Flavivirus members. Other receptors and associated pathways are used (i) alternatively or (ii) in addition. The results have important implications for further research on cellular factors that are crucial for WNV entry and thereby determine the basic mechanisms of infection. This knowledge would contribute to an improved understanding of WNV pathogenesis and of the susceptibility of cells in order to allow the control of this public health threat, and the prevention and treatment at the level of individuals, humans and animals, by the development of anti viral drugs targeting the virus entry.