Rabies virus(RABV) is a highly neurotropic virus that follows clathrin-mediated endocytosis and p H-dependent pathway for trafficking and invasion into endothelial cells. Early(Rab5, EEA1) and late(Rab7, LAMP1) endoso...Rabies virus(RABV) is a highly neurotropic virus that follows clathrin-mediated endocytosis and p H-dependent pathway for trafficking and invasion into endothelial cells. Early(Rab5, EEA1) and late(Rab7, LAMP1) endosomal proteins play critical roles in endosomal sorting, maturity and targeting various molecular cargoes, but their precise functions in the early stage of RABV neuronal infection remain elusive. In this study, the relationship between enigmatic entry of RABV with these endosomal proteins into neuronal and SH-SY5 Y cells was investigated.Immunofluorescence, TCID_(50) titers, electron microscopy and western blotting were carried out to determine the molecular interaction of the nucleoprotein(N) of RABV with early or late endosomal proteins in these cell lines. The expression of N was also determined by down-regulating Rab5 and Rab7 in both cell lines through RNA interference. The results were indicative that N proficiently colocalized with Rab5/EEA1 and Rab7/LAMP1 in both cell lines at 24 and 48 h post-infection, while N titers significantly decreased in early infection of RABV. Down-regulation of Rab5 and Rab7 did not inhibit N expression, but it prevented productive infection via blocking the normal trafficking of RABV in a low pH environment. Ultrathin sections of cells studied by electron microscope also verified the close association of RABV with Rab5 and Rab7 in neurons. From the data it was concluded that primary entry of RABV strongly correlates with the kinetics of Rab-proteins present on early and late vesicles, which provides helpful clues to explain the early events of RABV in nerve cells.展开更多
Rabies is caused by infection of rabies virus(RABV)and remains a serious threat to the global public health.Except for the requirement for cold chain and high cost of human rabies immune globulin,no small molecule dru...Rabies is caused by infection of rabies virus(RABV)and remains a serious threat to the global public health.Except for the requirement for cold chain and high cost of human rabies immune globulin,no small molecule drugs are currently available for clinical treatment of rabies.So,it is of great importance to identify novel compounds that can effectively inhibit RABV infection.Artesunate(ART)and dihydroartemisinin(DHA),two derivatives of artemisinin,are widely used for treatment of malaria in adults and children,showing high safety.In this study,we found that both ART and DHA were able to inhibit RABV replication in host cells at a low concentration(0.1μmol/L).The antiviral effects of ART and DHA were independent of viral strains and cell lines.Pre-treatment with ART or DHA for 2 h in vitro did not affect the viral replication in host cells,implying that ART and DHA neither reduced the viability of RABV directly nor inhibited the binding and entrance of the virus to host cells.Further studies revealed that ART and DHA inhibited RABV genomic RNA synthesis and viral gene transcription.Treatment with ART or DHA(5 mg/kg)by intramuscular injection improved,to some extent,the survival rate of RABV-challenged mice.Combination treatment with derivatives of artemisinin and mannitol significantly improved the survival rate of RABV-challenged mice.The results suggest that ART and DHA have a great potential to be explored as new anti-rabies agents for treatment of rabies.展开更多
While host proteins incorporated into virions during viral budding from infected cell are known to play essential roles in multiple process of the life cycle of progeny virus,these characteristics have been largely ne...While host proteins incorporated into virions during viral budding from infected cell are known to play essential roles in multiple process of the life cycle of progeny virus,these characteristics have been largely neglected in studies on rabies virus(RABV).Here,we purified the RABV virions with good purity and integrity,and analyzed their proteome by nano LC–MS/MS,followed by the confirmation with immunoblot and immuno-electronic microscopy.In addition to the 5 viral proteins,49 cellular proteins were reproducibly identified to be incorporated into matured RABV virions.Function annotation suggested that 24 of them were likely involved in virus replication.Furthermore,cryo-EM was employed to observe the purified RABV virions,generating high-resolution pictures of the bullet-shaped virion structure of RABV.This study has provided new insights into the host proteins composition in RABV virion and shed the light for further investigation on molecular mechanisms of RABV infection,as well as the discovery of new anti-RABV therapeutics.展开更多
基金supported by the National Key Research and Development Program of China(Grant No.216YFD0500402)Natural Science Foundation of China(Grants No.31272579 and 31472208)
文摘Rabies virus(RABV) is a highly neurotropic virus that follows clathrin-mediated endocytosis and p H-dependent pathway for trafficking and invasion into endothelial cells. Early(Rab5, EEA1) and late(Rab7, LAMP1) endosomal proteins play critical roles in endosomal sorting, maturity and targeting various molecular cargoes, but their precise functions in the early stage of RABV neuronal infection remain elusive. In this study, the relationship between enigmatic entry of RABV with these endosomal proteins into neuronal and SH-SY5 Y cells was investigated.Immunofluorescence, TCID_(50) titers, electron microscopy and western blotting were carried out to determine the molecular interaction of the nucleoprotein(N) of RABV with early or late endosomal proteins in these cell lines. The expression of N was also determined by down-regulating Rab5 and Rab7 in both cell lines through RNA interference. The results were indicative that N proficiently colocalized with Rab5/EEA1 and Rab7/LAMP1 in both cell lines at 24 and 48 h post-infection, while N titers significantly decreased in early infection of RABV. Down-regulation of Rab5 and Rab7 did not inhibit N expression, but it prevented productive infection via blocking the normal trafficking of RABV in a low pH environment. Ultrathin sections of cells studied by electron microscope also verified the close association of RABV with Rab5 and Rab7 in neurons. From the data it was concluded that primary entry of RABV strongly correlates with the kinetics of Rab-proteins present on early and late vesicles, which provides helpful clues to explain the early events of RABV in nerve cells.
基金We thank Dr.Xianzhu Xia(Academy of Military Medical Sciences)for providing the CVS-11 virus.We thank Dr.Zhenfang Fu(Huazhong Agricultural University)for providing the CVS-24 virus.This work was supported by the National Key Research and Development Program of China(2016YFD0500400)National Natural Science Foundation of China(31772742)Natural Science Foundation of Guangdong(2015A03031103).
文摘Rabies is caused by infection of rabies virus(RABV)and remains a serious threat to the global public health.Except for the requirement for cold chain and high cost of human rabies immune globulin,no small molecule drugs are currently available for clinical treatment of rabies.So,it is of great importance to identify novel compounds that can effectively inhibit RABV infection.Artesunate(ART)and dihydroartemisinin(DHA),two derivatives of artemisinin,are widely used for treatment of malaria in adults and children,showing high safety.In this study,we found that both ART and DHA were able to inhibit RABV replication in host cells at a low concentration(0.1μmol/L).The antiviral effects of ART and DHA were independent of viral strains and cell lines.Pre-treatment with ART or DHA for 2 h in vitro did not affect the viral replication in host cells,implying that ART and DHA neither reduced the viability of RABV directly nor inhibited the binding and entrance of the virus to host cells.Further studies revealed that ART and DHA inhibited RABV genomic RNA synthesis and viral gene transcription.Treatment with ART or DHA(5 mg/kg)by intramuscular injection improved,to some extent,the survival rate of RABV-challenged mice.Combination treatment with derivatives of artemisinin and mannitol significantly improved the survival rate of RABV-challenged mice.The results suggest that ART and DHA have a great potential to be explored as new anti-rabies agents for treatment of rabies.
基金This research was funded by the National Key Research and Development Plan(Grant No.2016YFD0500401)National Natural Science Foundation of China(Grant No.31402214)China Postdoctoral Science Foundation(Grant No.2014M552638).
文摘While host proteins incorporated into virions during viral budding from infected cell are known to play essential roles in multiple process of the life cycle of progeny virus,these characteristics have been largely neglected in studies on rabies virus(RABV).Here,we purified the RABV virions with good purity and integrity,and analyzed their proteome by nano LC–MS/MS,followed by the confirmation with immunoblot and immuno-electronic microscopy.In addition to the 5 viral proteins,49 cellular proteins were reproducibly identified to be incorporated into matured RABV virions.Function annotation suggested that 24 of them were likely involved in virus replication.Furthermore,cryo-EM was employed to observe the purified RABV virions,generating high-resolution pictures of the bullet-shaped virion structure of RABV.This study has provided new insights into the host proteins composition in RABV virion and shed the light for further investigation on molecular mechanisms of RABV infection,as well as the discovery of new anti-RABV therapeutics.
