| The life cycle of virus is usually belived to begin when virus attatches to the host cell membrane via its envelope proteins. The attatchment and following fusion of viral envelope proteins into cell membrane is thought to be very important for its successfully infecting host cells. Receptors on host cell membrane have the characters of specificity, high affinity, limited binding sites and other biological effects. Identifaction of the specific cellular receptors responsible for virus attachment is useful to understand tissue tropism, pathogenesis, and virus replication in cellular hosts, as well as to determine strategies for prevention and therapy of virus disease. Hantavirus (HV) infection is a major worldwide public health concern.Despite considerable advances in the understanding of natural history of HV disease, the process of virus replication and antigenic structure, most of the early steps in the virus life cycle remain unclear. Interfering with virus attachment to cell membrane is an important way for therapy. Recently, the research on HV cell membrane receptors concentrates mainly onβ3 integrins which may mediate the cellular entry of hantaviruses. However, no evidences show that there are direct interactions between hantavirus andβ3 integrin. Meanwhile, there are also some relative reports on other HV cell membrane co-receptors. But they require further research.To investigate the interaction of HV envelope G2 protein withβ3 intergrin and screen other putative receptors located on the cell surface to which HV binds, illuminate the mechanism of HV attachment to susceptible cells, EGFP-tagged fusion protein expression vectors encoding the envelope G2 fragments of HV 76-118 strain and Flag-taggedβ3 intergrin fragments expression vectors were constructed by PCR and gene recombination technology. Transient expression of these recombinant proteins in HEK293 cells was detected by Western blot. Only G2 N-terminal 81-140 fragment and allβ3 intergrin fragments were found in the supernatant of cell lysate. Pairs of the EGFP-tagged G2N81-140 and Flag-taggedβ3 intergrin fragments vectors were co-transfected into HEK293 cells and the interaction of envelope G2 protein withβ3 intergrin was identified by co-immunoprecipitation. The interaction between them could be identified by co-immunoprecipitation only in cells co-transfected with G2 81-140 andβ3 27-133 fragments expression vectors. GST-tagged G2N81-140 fusion protein expression vector was constructed and expressed in E.coli BL21 cells. The products were analyzed by SDS-PAGE and purified by Glutathione SepharoseTM4B. The supernatant of cell lysis containingβ3 intergrin 27-133 fragment was pre-cleared by GST protein and incubated with the purified G2N81-140 protein. The interaction of HV envelope G2 protein withβ3 intergrin was identified by GST pull-down. Our results show that hantavirus G2 may interact withβ3 intergrin and the interaction site may lie between G2 N-terminal 81-140 andβ3 intergrin 27-133 amino acids. Cell surface proteins of HV susceptible VeroE6 and non-permissive CHO cells were labeled by biotin. The supernatant of biotin-labeled cell lysis were pre-cleared by GST protein and incubated with the purified G2N81-140 protein. The cell surface proteins binding specifically to G2N81-140 were screened by GST pull down technique. Non-permissive CHO cells and unrelated protein GST-TLM were used as controls. A 30KDa protein on VeroE6 cell surface was found binding specifically to G2N81-140 fragment. Our result indicates that the 30KDa surface protein may be a putative receptor for HV 76-118 strain.Our results further show thatβ3 integrin may mediate the cellular entry of hantaviruses and there are also some other HV cell membrane co-receptors.
|
PDF Full Text Request