The Effect Of Clathrin And Endosome Acidification In Cells On Rotavirus Penetration

Rotaviruses are zoonotic agents, which infect children under five years of age or young animals and characterized by gastroenteritis with dehydration. Rotaviruses created great burden on society and economy, however therapeutic medicines specific for rotaviruses are unavailable now. The two live rotavirus vaccines are proven to be efficacious in the high-income developed countries,however the efficacy of both of two rotavirus vaccines is unsatisfied in the low-income countries. Thus, developing specific and effective drugs or efficient vaccines fighting against diseases due to rotavirus infection is imminent to solve the problem raised by rotaviruses. But how rotavirus enters into host cells remains to know. Study the mechanism of rotavirus entry into the host cell is necessary, which can provide a theoretical basis for the research of the drugs and vaccines for rotavirus.The open reading frame of VP6 of bovine rotavirus strain UK was expressed in prokaryotic cells and guniea pigs were immunized by the purified recombinant protein with the aid of Freund’s adjuvant. The serum was separated, then the VP6 polyclonal antibody was prepared and its character was analyzed. The MA104 or Caco-2 cells were treated with sucrose or NH4 Cl or untreated, the clathrin of selected cells was damaged or the endosome acidification of the cells was inhibited, followed by inoculation of UK or RRV rotaviruses. Whether the penetration of rotaviruses was inhibited or not upon analysis with indirect immunofluorescence. Meanwhile, the transcription of VP6 gene was quantitated by qRT-PCR to analyze the replication of UK or RRV rotaviruses.The VP6 polyclonal antibody was successfully prepared and reacted with UK strain and RRV strain rotaviruses. The number of fluorescent foci by UK rotaviruses group obviously decreased after the clathrin of cells was impaired by sucrose, however the that of RRV did not decrease obviously. As the result, the entry of the UK strain rotaviruses into the MA104 or Caco-2 cells depended on the clathrin, in contrast, the entry of RRV strain into MA104 or Caco-2 cells did not depend on the clathrin. The cells were treated by NH4 Cl and the endosome acidification of the cells was inhibited. It showed that the number of virus particles in UK group obviously decreased while the virus particles in RRV group did not change obviously. As the result, the entry of UK strain rotaviruses into the cells depended on the endosome acidification while RRV strain did not. In the real-time PCR, the VP6 of UK strain rotaviruses decreased after the Caco-2 cells and MA104 cells treated with sucrose and NH4 Cl, while the transcription of VP6 of RRV strainrotaviruses did not decrease obviously.The result upon Caco-2 cells was consistent with that upon MA104 cells. Above of all, both in the MA104 cells which are sensitive to rotaviruses and the Caco-2 cell which similar to the nature small intestine cells, the entry of UK strain rotaviruses into cells depended on the clathrin and the endosome acidification, the entry of RRV strain rotaviruses into the cells did not depend on the clathrin or the endosome acidification. The conclusion is important to clarify the mechanism of rotavirus entry. It also provides a reference to develop drugs to fight against gastroenteritis due to rotavirus infection.