Study On The Function And Application Of ZIKV Envelope Glycoprotein

Zika virus(ZIKV)Env protein is the main surface glycoprotein of virus.It plays an important role in the process of virus infection,adsorption and proliferation.This study focused on the ZIKV Env protein,mainly studies the biological function and application of Env protein.Clinical evidence indicates that there exists a strong connection between Zika virus(ZIKV)infection and abnormal development of the nervous system.However,the underlying mechanisms are still unclear.In the current study,recombinant lentiviral vectors of prM-Env,M-Env and Env of ZIKV were transduced into PC 12 cells.The envelope(Env)overexpression induced significant inhibition of proliferation and triggered G2/M cell cycle arrest and apoptosis in PC 12 cells.Flow cytometry and western blot analysis showed that the apoptosis was associated with up-regulation of both p53 and p21Cip1/Waf1 and down-regulation of cyclin B1.Presence of giant multinucleated cells was confirmed by immunofluorescence staining analysis.The data also indicate that overexpression of prM-Env,M-Env or Env led to apoptosis via an intrinsic cell death signaling pathway that is dependent on activation of caspase-9 and caspase-3,accompanied by an increased ratio of Bax to Bcl-2 in transduced PC 12 cells.Viral vectors including lentiviral vectors,adenoviral vectors and adeno-associated virus vectors are commonly utilized for gene delivery.Zika virus(ZIKV)infection is a mosquito-borne disease causing severe nervous system impairment including congenital microcephaly and Guillain-Barre syndrome.In this study,we designed and produced a pseudotype lentiviral vector bearing envelope glycoproteins(Env)of ZIKV.Infectivity of pseudotype lentivirus ZIKV-E generated in 293T cells can be inhibited by endosomal acidification inhibitors,indicating that ZIKA endocytosis in a pH-dependent manner.In vivo study indicated that ZIKV-E predominantly transduced the brain,heart and kidney of recipient mice.While it has extremely low transduction efficiency in the lung compared with VSV-G.More importantly,GFP fluorescence intensity assay indicated that transduction efficiency in the kidney with the ZIKV-E was ca.100-fold higher than that achieved with the VSV-G.Zikv virus(ZIKV)infection is one of the fastest spreading vector-borne diseases,and causes variable clinical symptoms,ranging from rash,fever,arthralgia and conjunctivitis to severe neurological disorders such as microcephaly and Guillain-Barre syndrome.In this study,we described the generation and immunogenicity of a mammalian cells-expressed ZIKV immunogen,ED?,based on ZIKV envelope protein domain ?.Vaccination with ED? elicited robust binding and neutralizing antibody responses in both BALB/c mice and macaques.After challenge with ZIKV,mice that received two doses of 10 ?g ED? subunit vaccine were largely protected from viremia with 10 of 12 animals having no detectable viremia.Two animals that received subunit vaccine had a low-level positive PCR on day 2,while naive animals had detectable viremia for 1-6 days post-challenge.Notably,peripheral blood mononuclear cell cultures from immunized macaques secreted high level of IL-2 and IL-6.Our data demonstrate that ED? is highly effective and represents a promising subunit vaccine candidate for ZIKV.In summary,the main achievements of the present study are as follow:In this study,we constructed several plasmids expression of the wild type ZIKV Env protein and the different deletion variants of Env protein,which laid the foundation for the subsequent biological research.Our data showed that ZIKV Env could induce apoptosis with the involvement of p53 via an intrinsic cell death signaling pathway in neuroendocrine PC 12 cells.This may indicate in the apoptosis induced by ZIKA Env in PC 12 cells,which may contribute to ZIKV-induced abnormal nervous system development.This study also reported the successful production of pseudotype lentiviral vector bearing envelope proteins of ZIKV for the first time.We further analyzed the infectious characteristics in BALB/c mice,results indicated that ZIKV-E has great potential in transduction targeting of renal cells.Furthermore,in vitro assay demonstrated that ZIKV-E entry cells through a pH-dependent manner,and a neutralization assay using ZIKV-E was developed for evaluating the neutralizing antibodies against ZIKV.Recombinant ED? proteins were successfully purified and their antigenicity was assessed.Immunization of mice and rhesus with recombinant proteins demonstrated the immunoprotection of the ED? protein.After mixed with MF59 adjuvant,subunit vaccine can produce high level cellular immune response in rhesus.Our results laid the foundation for the development of ZIKV genetic engineering subunit vaccine.